ATP-promoted interaction between Clp A and Clp P in activation of Clp protease from Escherichia coli

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Abstract
Summary Clp protease is a high relative molecular mass, ATP-dependent protease found in the cytoplasm of Escherichia coli. Clp protease is composed of two protein components, Clp A, which has ATPase activity, and Clp P, which has the proteolytic active site and is activated by Clp A in the presence of ATP. Clp P subunits (Mr = 21 500) are arranged in two hexagonal rings directly superimposed on each other, and under low salt conditions two dodecamers associate to form a particle with Mr ~ 440 000. Clp A (subunit Mr = 83 000) and Clp P do not associate in the absence of nucleotide, but Clp A with ATP bound associates with Clp P to form an active proteolytic complex with Mr ~ 700 000. Although adenosine 5′-[βγ-imido]triphosphate (AMPPNP) weakly promotes association between Clp A and Clp P, non-hydrolysable analogues of ATP do not activate proteolysis, indicating that association between the components is not sufficient to allow proteolysis. Association between Clp A and Clp P does not alter the basal ATPase activity of Clp A, but addition of protein substrates is accompanied by an increase in ATP hydrolysis by Clp A. Chemically-inactivated Clp P or inactive mutants of Clp P also associate with Clp A, but no increase in the ATPase activity of Clp A is observed, either in the presence or absence of protein substrates, when Clp P is inactive. Thus the increased ATP hydrolysis is dependent on active proteolysis. These data indicate that ATP has two functions in activating Clp protease: an allosteric role in promoting association between the subunits and a mechanistically undefined role in promoting continuous rounds of peptide bond cleavage. These functions may be separately fulfilled by the two ATP-binding sites identified by sequence analysis on each Clp A subunit.

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